[Radiation emergency medical preparedness in Japan--lessons learned from the Fukushima accident].

Although radiation exposure accidents fortunately occur only rarely, potential sources for exposure accidents can be found anywhere. When persons are accidentally exposed to radiation, physicians may be involved in their assessment and care; of course, their early diagnosis and dose assessment are crucial. After the criticality accident at Tokaimura in 1999, the system of radiation emergency medical preparedness has been further strengthened for nuclear facilities in Japan. In the revised system, hospitals involved were classified into three levels, depending on their locations and capabilities. The Great East Japan Earthquake attacked the Pacific coast area of eastern Japan on 11 March 2011. This earthquake and tsunami caused serious damage to the nuclear power plants of Tokyo Electric Power Co.(TEPCO) in Fukushima Prefecture; a large amount of radionuclides such as iodine and cesium were released into the environment. Since the revised system was focused on treatment of heavily exposed patients and knowledge on radiation was not enough for medical staff, many problems were raised at hospitals and fire departments in this disaster.

Genome wide screen identifies microsatellite markers associated with acute adverse effects following radiotherapy in cancer patients.

BACKGROUND: The response of normal tissues in cancer patients undergoing radiotherapy varies, possibly due to genetic differences underlying variation in radiosensitivity. METHODS: Cancer patients (n = 360) were selected retrospectively from the RadGenomics project. Adverse effects within 3 months of radiotherapy completion were graded using the National Cancer Institute Common Toxicity Criteria; high grade group were grade 3 or more (n = 180), low grade group were grade 1 or less (n = 180). Pooled genomic DNA (gDNA) (n = 90 from each group) was screened using 23,244 microsatellites. Markers with different inter-group frequencies (Fisher exact test P < 0.05) were analyzed using the remaining pooled gDNA. Silencing RNA treatment was performed in cultured normal human skin fibroblasts. RESULTS: Forty-seven markers had positive association values; including one in the SEMA3A promoter region (P = 1.24 x 10(-5)). SEMA3A knockdown enhanced radiation resistance. CONCLUSIONS: This study identified 47 putative radiosensitivity markers, and suggested a role for SEMA3A in radiosensitivity.

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level.

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method.

Visible Genotype Sensor Array.

A visible sensor array system for simultaneous multiple SNP genotyping has been developed using a new plastic base with specific surface chemistry. Discrimination of SNP alleles is carried out by an allele-specific extension reaction using immobilized oligonucleotide primers. The 3'-ends of oligonucleotide primers are modified with a locked nucleic acid to enhance their efficiency in allelic discrimination. Biotin-dUTPs included in the reaction mixture are selectively incorporated into extending primer sequences and are utilized as tags for alkaline phosphatase-mediated precipitation of colored chemical substrates onto the surface of the plastic base. The visible precipitates allow immediate inspection of typing results by the naked eye and easy recording by a digital camera equipped on a commercial mobile phone. Up to four individuals can be analyzed on a single sensor array and multiple sensor arrays can be handled in a single operation. All of the reactions can be performed within one hour using conventional laboratory instruments. This visible genotype sensor array is suitable for "focused genomics" that follows "comprehensive genomics".

Haplotype-based analysis of genes associated with risk of adverse skin reactions after radiotherapy in breast cancer patients.

PURPOSE: To identify haplotypes of single nucleotide polymorphism markers associated with the risk of early adverse skin reactions (EASRs) after radiotherapy in breast cancer patients. METHODS AND MATERIALS: DNA was sampled from 399 Japanese breast cancer patients who qualified for breast-conserving radiotherapy. Using the National Cancer Institute-Common Toxicity Criteria scoring system, version 2, the patients were grouped according to EASRs, defined as those occurring within 3 months of starting radiotherapy (Grade 1 or less, n = 290; Grade 2 or greater, n = 109). A total of 999 single nucleotide polymorphisms from 137 candidate genes for radiation susceptibility were genotyped, and the haplotype associations between groups were assessed. RESULTS: The global haplotype association analysis (p < 0.05 and false discovery rate < 0.05) indicated that estimated haplotypes in six loci were associated with EASR risk. A comparison of the risk haplotype with the most frequent haplotype in each locus showed haplotype GGTT in CD44 (odds ratio [OR] = 2.17; 95% confidence interval [CI], 1.07-4.43) resulted in a significantly greater EASR risk. Five haplotypes, CG in MAD2L2 (OR = 0.55; 95% CI, 0.35-0.87), GTTG in PTTG1 (OR = 0.48; 95% CI, 0.24-0.96), TCC (OR = 0.48; 95% CI, 0.26-0.89) and CCG (OR = 0.50; 95% CI, 0.27-0.92) in RAD9A, and GCT in LIG3 (OR = 0.46; 95% CI, 0.22-0.93) were associated with a reduced EASR risk. No significant risk haplotype was observed in REV3L. CONCLUSION: Individual radiosensitivity can be partly determined by these haplotypes in multiple loci. Our findings may lead to a better understanding of the mechanisms underlying the genetic variation in radiation sensitivity and resistance among breast cancer patients.

Gene expression profile changes correlating with radioresistance in human cell lines.

PURPOSE: To identify gene expression profiles specific to radioresistance of human cells. METHODS AND MATERIALS: Global gene expression profiles of a total of 15 tumor and normal fibroblast cell lines were analyzed using DNA microarrays and statistical clustering methods. Initially, six of the cell lines were categorized into radioresistant (RG) or nonradioresistant (NRG) groups according to the radiation dose required to reduce their survival to 10% (D10). Genes for which expression was specific to each group at 1 or 3 h after irradiation were identified using statistical procedures including analysis of variance and a two-dimensional hierarchical clustering method. The remaining nine cell lines were subjected to the k-nearest neighbor pattern classification. RESULTS: The nine test cell lines were successfully classified by their D10 value using 46 and 44 genes for which transcription levels had significantly changed at 1 and 3 h after irradiation, respectively. Of these genes, 25 showed altered expression at both time points in the NRG or RG, but independently were unable to classify the test cell lines. CONCLUSIONS: Radioresistant cell lines analyzed in this study showed certain radiation-induced changes in gene expression profiles that are different from the profile changes of the more-sensitive cell lines.

Reliable and fast allele-specific extension of 3'-LNA modified oligonucleotides covalently immobilized on a plastic base, combined with biotin-dUTP mediated optical detection.

In the present work, a convenient microarray SNP typing system has been developed using a plastic base that covalently immobilizes amino-modified oligonucleotides. Reliable SNP allele discrimination was achieved by using allelic specificity-enhanced enzymatic extension of immobilized oligonucleotide primer, with a locked nucleic acid (LNA) modification at the SNP-discriminating 3'-end nucleotide. Incorporation of multiple biotin-dUTP molecules during primer extension, followed by binding of alkaline phosphatase-conjugated streptavidin, allowed optical detection of the genotyping results through precipitation of colored alkaline phosphatase substrates onto the surface of the plastic base. Notably, rapid primer extension was demonstrated without a preliminary annealing step of double-stranded template DNA, allowing overall processes to be performed within a couple of hours. Simultaneous evaluation of three SNPs in the genes TGFB1, SOD2 and APEX1, previously investigated for association with radiation sensitivity, in 25 individuals has shown perfect assignment with data obtained by another established technique (MassARRAY system).

Vitronectin improves cell survival after radiation injury in human umbilical vein endothelial cells.

Vitronectin (VN) is a multi-functional protein involved in extracellular matrix (ECM)-cell binding through integrin receptors on the cell surface, which is an important environmental process for maintaining biological homeostasis. We investigated how VN affects the survival of endothelial cells after radiation damage. VN attenuated radiation-induced expression of p21, an inhibitor of cell cycle progression, and selectively inhibited Erk- and p38 MAPK-dependent p21 induction after radiation exposure through regulation of the activity of GSK-3ß. VN also reduced the cleavage of caspase-3, thereby inhibiting radiation-induced apoptotic cell death. These results suggest that VN has important roles in cell survival after radiation damage.

Dynamics of delayed p53 mutations in mice given whole-body irradiation at 8 weeks.

PURPOSE: Ionizing irradiation might induce delayed genotoxic effects in a p53-dependent manner. However, a few reports have shown a p53 mutation as a delayed effect of radiation. In this study, we investigated the p53 gene mutation by the translocation frequency in chromosome 11, loss of p53 alleles, p53 gene methylation, p53 nucleotide sequence, and p53 protein expression/phosphorylation in p53(+/+) and p53(+/-) mice after irradiation at a young age. METHODS AND MATERIALS: p53(+/+) and p53(+/-) mice were exposed to 3 Gy of whole-body irradiation at 8 weeks of age. Chromosome instability was evaluated by fluorescence in situ hybridization analysis. p53 allele loss was evaluated by polymerase chain reaction, and p53 methylation was evaluated by methylation-specific polymerase chain reaction. p53 sequence analysis was performed. p53 protein expression was evaluated by Western blotting. RESULTS: The translocation frequency in chromosome 11 showed a delayed increase after irradiation. In old irradiated mice, the number of mice that showed p53 allele loss and p53 methylation increased compared to these numbers in old non-irradiated mice. In two old irradiated p53(+/-) mice, the p53 sequence showed heteromutation. In old irradiated mice, the p53 and phospho-p53 protein expressions decreased compared to old non-irradiated mice. CONCLUSION: We concluded that irradiation at a young age induced delayed p53 mutations and p53 protein suppression.

Combination effects of distinct cores in 11q13 amplification region on cervical lymph node metastasis of oral squamous cell carcinoma.

Lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC) is known to associate with a significant decrease of 5-year survival. Genetic factors related to the difference of the LNM status in the OSCC have been not fully elucidated. Array-based comparative genomic hybridization (CGH) with individual gene-level resolution and real-time quantitative polymerase chain reaction (QPCR) were conducted using primary tumor materials resected from 54 OSCC patients with (n=22) or without (n=32) cervical LNM. Frequent gain was observed at the 11q13 region exclusively in patients with cervical LNM, which was confirmed by real-time QPCR experiments using 11 genes (TPCN2, MYEOV, CCND1, ORAOV1, FGF4, TMEM16A, FADD, PPFIA1, CTTN, SHANK2 and DHCR7) in this region. It was revealed that two distinct amplification cores existed, which were separated by a breakpoint between MYEOV and CCND1 in the 11q13 region. The combination of copy number amplification at CTTN (core 2) and/or TPCN2/MYEOV (core 1), selected from each core, was most significantly associated with cervical LNM (P=0.0035). Two amplification cores at the 11q13 region may have biological impacts on OSCC cells to spread from the primary site to local lymph nodes. Further study of a larger patient series should be conducted to validate these results.

Genetic variants of NPAT-ATM and AURKA are associated with an early adverse reaction in the gastrointestinal tract of patients with cervical cancer treated with pelvic radiation therapy.

PURPOSE: This study sought to associate polymorphisms in genes related to cell cycle regulation or genome maintenance with radiotherapy (RT)-induced an early adverse reaction (EAR) in patients with cervical cancer. METHODS AND MATERIALS: This study enrolled 243 cervical cancer patients who were treated with pelvic RT. An early gastrointestinal reaction was graded using the National Cancer Institute Common Toxicity Criteria, version 2. Clinical factors of the enrolled patients were analyzed, and 208 patients were grouped for genetic analysis according to their EAR (Grade ≤1, n = 150; Grade ≥2, n = 58). Genomic DNA was genotyped, and association with the risk of EAR for 44 functional single-nucleotide polymorphisms (SNPs) of 19 candidate genes was assessed by single-locus, haplotype, and multilocus analyses. RESULTS: Our analysis revealed two haplotypes to be associated with an increased risk of EAR. The first, comprising rs625120C, rs189037T, rs228589A, and rs183460G, is located between the 5' ends of NPAT and ATM (OR = 1.86; 95% CI, 1.21-2.87), whereas the second is located in the AURKA gene and comprises rs2273535A and rs1047972G (OR = 1.75; 95% CI, 1.10-2.78). A third haplotype, rs2273535T and rs1047972A in AURKA, was associated with a reduced EAR risk (OR = 0.42; 95% CI, 0.20-0.89). The risk of EAR was significantly higher among patients with both risk diplotypes than in those possessing the other diplotypes (OR = 3.24; 95% CI, 1.52-6.92). CONCLUSIONS: Individual radiosensitivity of intestine may be determined by haplotypes in the NPAT-ATM and AURKA genes. These variants should be explored in larger association studies in cervical cancer patients.

Villin1, a novel diagnostic marker for cervical adenocarcinoma.

The number of new cervical adenocarcinoma (AD) cases has risen slowly, however, its histological similarity to other tumor types and the difficulty of identifying the site of the original tumor makes the diagnosis of cervical AD particularly challenging. We investigated a novel molecular biomarker for cervical AD through the integration of multiple methods of genomic analysis. Tumor samples in discovery set were obtained from 87 patients who underwent radiotherapy, including 31 cervical AD. Microarray analysis and quantitative polymerase chain reaction analysis were performed to screen a candidate diagnostic molecule for cervical AD, and its clinical significance was investigated by immunohistochemical analysis (IHC). We found a difference between biopsy samples of AD and squamous cell carcinoma (SCC) in the expression and genomic copy number of Villin1 (VIL1), which maps to 2q35. IHC revealed 14 VIL1-positive tumors; 13 cervical AD and one small cell carcinoma of cervix, while none of SCC or endometrial AD was VIL1-positive. Kaplan-Meier survival curves revealed worse disease-free survival in VIL1-positive tumors. The marker was validated by newly enrolled 65 patients, and VIL1 positive staining showed 52% of sensitivity and 100% of selectivity for cervical AD. In conclusion, we have identified VIL1 as a novel biomarker of cervical AD. VIL1, a major structural component of the brush border cytoskeleton, which was recently found to be an epithelial cell-specific anti-apoptotic protein. Our study suggests the existence of a subtype of cervical tumors which are VIL1 positive with poor radioresponse.

Radiation sensitivities of 31 human oesophageal squamous cell carcinoma cell lines.

The purpose of this study was to determine the radiosensitivities of 31 human oesophageal squamous cell carcinoma cell lines with a colony-formation assay. A large variation in radiosensitivity existed among 31 cell lines. Such a large variation may partly explain the poor result of radiotherapy for this cancer. One cell line (KYSE190) demonstrated an unusual radiosensitivity. Ataxia-telangiectasia-mutated (ATM) gene in these cells had five missense mutations, and ATM protein was truncated or degraded. Inability to phosphorylate Chk2 in the irradiated KYSE190 cells suggests that the ATM protein in these cells had lost its function. The dysfunctional ATM protein may be a main cause of unusual radiosensitivity of KYSE190 cells. Because the donor of these cells was not diagnosed with ataxia telangiectasia, mutations in ATM gene might have occurred during the initiation and progression of cancer. Radiosensitive cancer developed in non-hereditary diseased patients must be a good target for radiotherapy.

Strikingly higher frequency in centenarians and twins of mtDNA mutation causing remodeling of replication origin in leukocytes.

The presence of a genetic component in longevity is well known. Here, the association of a mtDNA mutation with a prolonged life span in humans was investigated. Large-scale screening of the mtDNA main control region in leukocytes from subjects of an Italian population revealed a homoplasmic C150T transition near an origin of heavy mtDNA-strand synthesis in approximately 17% of 52 subjects 99-106 years old, but, in contrast, in only 3.4% of 117 younger individuals (P = 0.0035). Evidence was obtained for the contribution of somatic events, under probable nuclear genetic control, to the striking selective accumulation of the mutation in centenarians. In another study, among leukocyte mtDNA samples from 20 monozygotic and 18 dizygotic twins, 60-75 years old, 30% (P = 0.0007) and 22% (P = 0.011), respectively, of the individuals involved exhibited the homoplasmic C150T mutation. In a different system, i.e., in five human fibroblast longitudinal studies, convincing evidence for the aging-related somatic expansion of the C150T mutation, up to homoplasmy, was obtained. Most significantly, 5' end analysis of nascent heavy mtDNA strands consistently revealed a new replication origin at position 149, substituting for that at 151, only in C150T mutation-carrying samples of fibroblasts or immortalized lymphocytes. Considering the aging-related health risks that the centenarians have survived and the developmental risks of twin gestations, it is proposed that selection for a remodeled replication origin, inherited or somatically acquired, provides a survival advantage and underlies the observed high incidence of the C150T mutation in centenarians and twins.

[RadGenomics project].

Human health conditions are largely determined by a complex interplay among genetic susceptibility, environmental factors, and aging. The RadGenomics project, which began in April 2001, promotes analysis of genes in response to irradiation, identification of their allelic variants in the human population, development of an effective procedure for quantitating individual radio-sensitivity, and analysis of the interrelationship between genetic heterogeneity and susceptibility to irradiation. Major groups of genes with which the project will concern itself include DNA repair genes, cell cycle genes, oncogenes, tumor suppressor genes, genes for programmed cell death, genes for signal transduction, and genes for oxidative processes. The outcome of the RadGenomics project should lead to improved protocols for personalized radiotherapy and reduce the possible side effects of treatment. The project will contribute to future research on the molecular mechanisms of radiation sensitivity in humans and stimulate the development of new high-throughput technology for a broader application of the biological and medical sciences. Identification of functionally important polymorphisms in the radiation response genes may determine individual differences in sensitivity to radiation exposure. The staff members, who are specialists in a variety of fields including genome science, radiation biology, medical science, molecular biology, and bioinformatics, have come to the RadGenomics project from various universities, companies, and research institutes.